This project is concerned with defining mechanisms by which substances are transported into and out of cells. The experiments will make use of microorganisms (Salmonella typhimurium) which have genetic mutations in their transport systems, and which will be analyzed biochemically for the missing factors. We already isolated a variety of such mutants defective in various steps of the histidine transport systems, and more different types will be sought. One essential component of histidine transport has already been identified by use of these mutants: the histidine-binding protein J, coded for by the hisJ gene. A search for another component, the P protein (coded for by the hisP gene) is under way. Methods for the solubilization of membranes and for a resolution of its components by two-dimensional gel electrophoresis have been developed. The relationship between the J protein and the P protein, their mode of function and their direct interaction with each other are being analyzed by a variety of biochemical techniques: e.g., the direct binding of the J protein to the P protein; the effect of the P protein upon the affinity of histidine for the J protein; the effect of possible energy-supplying compounds (e.g., ATP, PEP, D-lactate) upon a J-P protein interaction; reconstitution of transport in partially purified systems (vesicles, liposomes, spheroplasts) as detected by recovery of transport or by binding of labelled transport components to membrane surfaces; cross-linking of the histidine-binding protein J to the P protein in vivo; biochemical studies on the nature of the two active sites known to exist in the J protein.